The major objectives of this project are to determine the detailed mechanism of replication of single-stranded RNA viral genomes and to analyze the factors which control viral RNA replication. The viruses used for this project are two human pathogens, poliovirus and influenza virus. Virus-specific ribonucleoprotein structures (RNPs) will be isolated from the nucleus and cytoplasm of cells infected with influenza virus. These RNPs function in RNA replication and protein synthesis. The biochemical composition, enzymatic function, assembly and distribution of these RNPs will be studied. The role of host cell function in the replication of influenza virus will be studied by analysis of the nuclear phase of viral replication and by characterization of the polypeptides present in the nucleus after infection. The effect of inhibitors such as dichlorobenzimidazole riboside (DRB) and actinomycin D on plus-stranded and minus-stranded influenza virus-specific RNA synthesis in the nucleus and cytoplasm of infected cells will be studied. Virus-specific structures involved in RNA synthesis will be isolated from poliovirus-infected cells and the biochemical, biophysical and enzymatic properties of these structures will be characterized. In vitro assay systems to study RNA synthesis in these structures will be developed using non-denaturing polyacrylamide gel electrophoresis. The role of cytoplasmic membranes in RNA and protein synthesis and processing will be determined. Procedures for the further purification of a template-dependent RNA replicase from poliovirus-infected cells will be developed. These studies will lead to a better understanding of the detailed mechanism of RNA synthesis, the regulation of this process, and the role of host cell factors in viral replication. This knowledge should lead to a rational approach to the control of viral replication by selective inhibitors, a better understanding of virus-cell interactions, and a model for the biogenesis and function of RNP structures in normal and infected eucaryotic cells. BIBLIOGRAPHIC REFERENCES: Caliguiri, L.A. and Gerstein, H. Isolation and characterization of RNA-protein complexes from influenza virus-infected cells. Fed. Proc. (Abstract) (1976) in press.